Engineered, clinically-relevant production of helper (cd4+) t cells from pluripotent stem cells

Background & Aim Engineered T cells have great potential for treating cancer, chronic viral infections, and autoimmunity. However, current autologous T cell therapies are limited by high costs and heterogeneity. Producing allogeneic ‘off-the-shelf’ T cells from pluripotent stem cells (PSCs) could dramatically reduce costs, increase access to therapy, and unlock advanced cell engineering. Recently, we developed a clinically-relevant feeder- and serum-free method for producing cytotoxic (CD8+) T cells from PSCs (PMID: 36001668). However, production of helper (CD4+) T cells remains a challenge in the field. As CD4+ T cells are capable of diverse and critical immune functions including improving anti-cancer potency of CD8+ T cells, we aimed to produce CD4+ T cells from PSCs in vitro. Methods, Results & Conclusion We used design-of-experiments and response surface modeling (RSM) to identify and optimize the levels of cytokines that enrich for committed CD4+/CD8+ double positive (DP), CD4+/CD8- (4SP), or CD4-/CD8+ (8SP) cells in feeder- and serum-free conditions (Fig. 1A). We then systematically tested combinations and timings of optimal media to induce production of committed (CD27+) 8SP and 4SPs (Fig. 1B). From these experiments, we identified conditions to increase yields of both 8SPs and 4SPs by 1-2 orders of magnitude over our published protocol. Further improvements have enabled robust production of mature, committed 4SPs from PSCs, with initial yields >1 4SP per CD34+ cell input, and the ability to expand >10-fold. Post-expansion, our PSC-4SPs show a highly mature phenotype, expressing late maturation/naïve CD4+ T cell markers including CD62L and CD40L (Fig. 1C). We further confirmed that our PSC-4SPs do not express 8SP-associated cytotoxic genes including granzyme B and perforin. Overall, we identified feeder- and serum-free conditions to produce mature 4SPs from PSCs, opening the door for the development of new allogeneic, PSC-derived therapies utilizing helper T cells. Our platform will also support efforts to build immune-competent organoid and tissue models by providing an isogenic and renewable source of diverse types of T cells. Funding Wellcome Leap HOPE Program, Health Research BC Postdoctoral Fellowship.